title: Cytoskeletal alterations in different developmental stages of in vivo cryopreserved preimplantation murine embryos.
abstract: BACKGROUND
  This study aimed to investigate the effects of vitrification and slow freezing on actin, tubulin, and nuclei of in vivo preimplantation murine embryos at various developmental stages using a Confocal Laser Scanning Microscope (CLSM).
  
  
  METHODS
  Fifty female mice, aged 4-6 weeks, were used in this study. Animals were superovulated, cohabitated overnight, and sacrificed. Fallopian tubes were excised and flushed. Embryos at the 2-cell stage were collected and cultured to obtain 4- and 8-cell stages before being cryopreserved using vitrification and slow freezing. Fixed embryos were stained with fluorescence-labelled antibodies against actin and tubulin, as well as DAPI for staining the nucleus. Labelled embryos were scanned using CLSM and images were analyzed with Q-Win software V3.
  
  
  RESULTS
  The fluorescence intensity of both vitrified and slow-frozen embryos was significantly lower for tubulin, actin, and nucleus as compared to non-cryopreserved embryos (p<0.001). Intensities of tubulin, actin, and nucleus in each stage were also decreased in vitrified and slow-frozen groups as compared to non-cryopreserved embryos.
  
  
  CONCLUSIONS
  Cryopreservation of mouse embryos by slow freezing had a more detrimental effect on the actin, tubulin, and nucleus structure of the embryos compared to vitrification. Vitrification is therefore superior to slow freezing in terms of embryonic cryotolerance.
authors.affiliation: Institute of Medical Molecular Biotechnology, Faculty of Medicine, Universiti Teknologi MARA, Selangor, Malaysia and Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia.
authors.forename: Razif
authors.initials: R
authors.lastname: Dasiman
authors.forename: Nor-Shahida Abdul
authors.initials: NS
authors.lastname: Rahman
authors.forename: Salina
authors.initials: S
authors.lastname: Othman
authors.forename: Mohd-Fazirul
authors.initials: MF
authors.lastname: Mustafa
authors.forename: Norhazlin Jusoh Mohd
authors.initials: NJ
authors.lastname: Yusoff
authors.forename: Wan-Hafizah W
authors.initials: WH
authors.lastname: Jusoff
authors.forename: Mohd Hamim
authors.initials: MH
authors.lastname: Rajikin
authors.forename: Gabriele Ruth Anisah
authors.initials: GR
authors.lastname: Froemming
authors.forename: Nor-Ashikin Mohamed Noor
authors.initials: NA
authors.lastname: Khan
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D015925:Cryopreservation; D003599:Cytoskeleton / Q000378:metabolism*; D047108:Embryonic Development; D005260:Female; D005453:Fluorescence; D051379:Mice; D008813:Mice, Inbred ICR; D018613:Microscopy, Confocal; D014404:Tubulin / Q000378:metabolism
chemical_list: D000199:Actins; D014404:Tubulin